Phenylephrine-induced cardiomyocyte injury is triggered by superoxide generation through uncoupled endothelial nitric-oxide synthase and ameliorated by 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836), a novel calmodulin antagonist.
The pathophysiological relevance of endothelial nitric-oxide synthase (eNOS)-induced superoxide production in cardiomyocyte injury after prolonged phenylephrine (PE) exposure remains unclear. The aims of this study were to define the mechanism of O2(*) production by uncoupled eNOS and evaluate the therapeutic potential of a novel calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836) to rescue hypertrophied cardiomyocytes from PE-induced injury. In cultured rat cardiomyocytes, prolonged exposure for 96 h to PE led to translocation from membrane to cytosol of eNOS and breakdown of caveolin-3 and dystrophin. When NO and O2(*) production were monitored in PE-treated cells by 4-amino-5-methylamino-2',7'-difluorofluorescein and dihydroethidium, respectively, Ca(2+)-induced NO production elevated by 5.7-fold (p < 0.01) after 48-h PE treatment, and the basal NO concentration markedly elevated (16-fold; p < 0.01) after 96-h PE treatment. On the other hand, the O2(*) generation at 96 h was closely associated with an increased uncoupled eNOS level. Coincubation with DY-9836 (3 microM) during the last 48 h inhibited the aberrant O2(*) generation nearly completely and NO production by 72% (p < 0.01) after 96 h of PE treatment and inhibited the breakdown of caveolin-3/dystrophin in cardiomyocytes. PE-induced apoptosis assessed by TdT-mediated dUTP nick-end labeling staining was also attenuated by DY-9836 treatment. These results suggest that O2(*) generation by uncoupled eNOS probably triggers PE-induced cardiomyocyte injury. Inhibition of abnormal O2(*) and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.
Lu
Ying-Mei
YM
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Han
Feng
F
Shioda
Norifumi
N
Moriguchi
Shigeki
S
Shirasaki
Yasufumi
Y
Qin
Zheng-Hong
ZH
Fukunaga
Kohji
K
D000818:Animals; D000831:Animals, Newborn; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002316:Cardiotonic Agents / Q000494:pharmacology*; D051244:Caveolin 3 / Q000378:metabolism; D002478:Cells, Cultured; D016189:Dystrophin / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology; D007150:Immunohistochemistry; D007191:Indazoles / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010590:Phalloidine / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines / Q000378:metabolism; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors
C534583:3-(2-(4-(3-chloro-2-methylphenyl)-1-piperazinyl)ethyl)-5,6-dimethoxyindazole; D002147:Calmodulin; D002316:Cardiotonic Agents; D051244:Caveolin 3; D016189:Dystrophin; D005456:Fluorescent Dyes; D007191:Indazoles; D010879:Piperazines; D012235:Rhodamines; D013481:Superoxides; D010590:Phalloidine; D010656:Phenylephrine; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III